Patented Oct. 9, 1951 2,570,297 UNITED STATES PATENT OFFICE 2,570,297 AMNO ACD SYNTHESIS David I. Weisblat and Douglas A. Lyttle, Kala mazoo, Mich., assignors to The Upjohn Com pany, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application November 29, 1946, Serial No. 713,094 3 Claims. (CI. 260-534) 1. 2 The present invention relates to the prepara (1928)). Because of the difficulty of synthesis, tion of amino acids and is more particularly the preferred method of obtaining this amino concerned with a novel method for the Synthesis acid is by the hydrolysis of gelatin (Blatt Organic of alpha amino acids. Syntheses, col. vol. II, 59-62, John Wiley and The significance of the alpha amino acids in Sons, New York, 1943). Histidine has been pre human and animal nutrition makes their Syn pared by procedures 2, 3, (a) and 4 (c). Waline thesis generally of great importance. The volumi (Organic Syntheses 20, 106 (1940)), leucine, and nous literature and the great number of attempts isoleucine are best prepared by methods 1 and 3 to find suitable procedures for the preparation of (a). Phenylalanine has been prepared by meth alpha primary amino acids is indicative of this 0. ods 3 (b), 4 (b) and 4 (c). (Blatt Org. Syn., importance. (Gilman, Organic Chemistry, vol.II, col. vol. II, 489-94, John Wiley and Sons, New "Natural amino acids,' by H. T. Clark, pp. 1079 York, 1943.) Threonine has been prepared by a 1166, John Wiley and Sons, New York, 1943; procedure under method 2 (Org. Syn. 20, 101, Schmidt, “The Chemistry of Amino Acids and John Wiley and Sons, New York, 1940), the neces Proteins," chapter II, "The Constitution and syn 5 Sary halogen acid being obtained from crotonic thesis of amino acids' by Max S. Dunn, Charles acid. C. Thomas, Baltimore, 1938; Wickery and Schmidt, Other amino acids which are not considered Chem. Rev. 9, 169-318 (1931)). The principal as essential to certain animal diets have been methods which previously have been employed prepared by variations of these methods. They for the preparation of amino acids involve: i. 20 are aspartic acid, glutamic acid, hydroxyglutamic The cyanohydrin synthesis (Strecker synthesis). acid, glycine, alanine, norieucine, serine, cysteine 2. The action of ammonia on alpha halogen acids. and tyrosine. 3.Reactions using malonic esters (a) to give alpha Inasmuch as the amino acids appear to play an halogen acids for method (2), (b) reaction of increasingly important role in the nutrition and phthalamidomalonic ester with halogen Com 25 physiology of the human race, the importance pounds, (c) reactions of amino malonic esters, thereof, as well as the importance of a new and (d) reaction of potassium ethylmalonates with more practicable synthesis therefor, is considered hydrazine. 4. Condensation of aldehydes with (a) obvious. hydantoin, (b) diketOpiperazine, (c) hippuric It is an object of the present invention to acid (azlactone method). 5. From alpha, keto provide a new method for the synthesis of amino acids by (a) reduction and amination, (b) re acids. An additional object of the invention is duction of oximes, (c) reduction of hydrazones. the provision of a method for the synthesis of Detailed procedures for the production of alpha amino acids in a manner which may be conducted amino acids usually involve, in Some stage of the With increased facility in comparison with known synthesis, one of these general methods. The 35 methods. A further object of the invention is the preparation of tryptophane from gramine (Al provision of a novel method for the synthesis bertson et al., J. Am. Chem. Soc. 66, 500 (1944), of amino acids which will be more economically 67, 36-7 (1945); Snyder et al., J. Am. Chem. Soc. and commercially practicable. Another object 66, 350 (1944); Howe et al., J. Am. Chem. Soc. of the invention is the provision of a novel method 67, 38 (1945)) may be considered as a variant of 40 for the production of amino acids which in method 3 (b). The preparation of methionine volves the alkylation of an ester of nitroacetic (Windus and Marvel, J. Am. Chem. Soc. 52, 2575 acid. A further object of the invention is the (1930); Barger and Weichselbaum in Blatt Org. provision of a process for the preparation of Synthesis, col vol. II, 384, John Wiley and Sons, anino acids which includes the step of reducing New York, (1943) proceeds through malonic ester an alpha nitro acid or an ester thereof. Another or phthalimidomalonic ester by procedures 3 (a) object of the invention is the provision of a and 3 (b). Ornithine, necessary for the prepar method for the Synthesis of amino acids which ation of arginine, has been prepared by method 3 proceeds through nitro acids or nitro acid esters (b) (Fischer, Ber. 34,454 (1901)), as the dipicrate corresponding to the desired amino acid. A fur from CH2=CH-CH=CH-COOH (beta vinyla ther object of the invention is the provision of a crylic acid) and ammonia (Fischer and Raske, novel method for the synthesis of amino acids Ber. 38, 3607 (1905)), and as the dibenzoyl de which may be conducted in three steps from the rivative, by Kumatsu and Sugasawa, following starting nitroacetate, or with various combina an eleven step procedure from acrolein (J. Pharm. tions of steps when starting with an intermediate. Soc., Japan, 48, 24 (1929); Chem Abstr. 28, 1758 Another object of the invention is the provision 2,570,29? 3 4. of a method for the preparation of amino acid compounds have been found especially satisfaca which proceeds through the steps of alkylation, tory, and their employment in the alkylation reduction, hydrolysis and, if desired, resolution. step therefore constitutes a preferred embodil Another object of the invention is the provision ment of the invention. of a process which involves the alkylation of an The alkylation step of our new synthesis may, ester of nitroacetic acid, reduction and hydrolysis for example, be carried out by reacting together of the resulting nitro acid ester to the amino an ester of nitroacetic acid, e. g., ethyl nitro acid. Another object of the invention is the pro acetate, and a suitable alkylating agent, for ex vision of a process for the preparation of Optical ample, One Such as is utilised in the preparation isomers of amino acids in substantially pure 10 of tryptophane, gramine (3 - dimethylamino form. A still further object of the invention is methylindole), with or without the employment the provision cf novel compounds, intermediates Of alkali and heat. We have found that when in the preparation of amino acids, including the process is conducted in this manner, e. g., esters of nitro acids, the nitro acids themselves, with the employment of a nitroacetate and a and, in some cases, the esters of the amino acids. 5 Substituted dialkylamine such as gramine, it is Other objects of the invention will be apparent usually advantageously, although not necessarily, from the following specification and claims. carried out in an anhydrous organic solvent such . The method of our invention is generally ap as xylene, while passing a slow stream of nitro plicable to the preparation of the above-named gen through the reaction with vigorous agitation, and other primary alpha amino acids. The 20 at a temperature usually up to about 100 degrees method of our invention essentially involves the centigrade over a period of five hours, more or treatment of an ester of nitroacetic acid, e. g., less. During this period of heating, a consider ethyl nitroacetate, with a selected "alkylating able quantity of dialkylamine (dimethylamine agent," i. e., an agent of the alkylating type from gramine) is evolved. The hot solution may which is capable of introducing a desired group 25 then be filtered from a small amount of crystal into the acid portion of the nitroacetate mole line solid which usually forms and the xylene cule at the alpha carbon atom, reduction of the removed by concentration in vacuo. The residual nitro ester thus formed, isolation of the amino gun may be dissolved in chloroform, the solu ester and separation of the same into its optical tion extracted with dilute hydrochloric acid and enantiomorphs if desired, and hydrolysis of the 30 washed with water until neutral. The remain amino ester to the particular amino acid con ing solution may be dried, concentrated in a cerned. vacuum, freed of excess nitroacetate by distilla The alkylation step. of our new Synthesis may tion under reduced pressure, dissolved in chloro be carried out by reacting together any desired form. Or similar Solvent, and extracted to ex ester of nitroacetic acid, or salts thereof, and a 35 haustion with dilute alkali. After acidification Selected "alkylating agent.' By "alkylating and extraction with chloroform or similar Sol agent,' as herein employed, is intended any vent, drying and concentrating the last chloro agent comprising at least a selected group, and form extract usually leaves an oil which may capable of introducing said group into the acid be crystallised readily. This oil is an ester of portion of the nitroacetate molecule at the alpha 40 the desired substituted nitroacetic acid, in the carbon atom. As agents in this capacity may case of the foregoing example wherein gramine and ethyl nitroacetate were reacted, of alpha be mentioned: nitro-beta- (3-indole) propionic acid, in a sub 1A. Alkyl halides, alkyl arylsulfonates, and stantially pure form. Other methods may be alkyl sulfates with a basic catalysis; m employed to separate the ester and will be ap B. Carbinols with boron trifluoride catalysis; 45 parent to One skilled in the art. C. Substituted dialkylamines (tertiary amines), The above alkylation of a nitroacetic acid ester Or quaternary ammonium compounds with or with a substituted dialkylamine is exemplary of without alkali and/or heat; the type of alkylation hereinafter described as 2. Aldehydes or ketones with acidic, basic, or type 1. The type of alkylation hereinafter de neutral catalysis according to Knoevenagel type 50 scribed as type 2 is effected by the use of an alde of condensation. hyde or ketone with an acidic, basic, or neutral 3A. Compounds containing activated double catalysis, and is usually, although not necessarily, bonds; conducted at a relatively low temperature, in the B. Compounds containing epoxide or thioepox presence of a Catalyst Such as piperidine or para ide Systems. 55 toluenesulfonic acid. Either an alkylidene nitro It will be noted that the esters of nitroacetic ester or a hydroxy-containing nitro ester is the acid, NO2-CH2-COOR, may now be considered first product of the reaction, and if it is not de as being related to malonic and cyanacetic esters, sired to produce an amino acid containing a ROOC-CH-COOR and NC-CH2-COOR, re hydroxyl group On the beta carbon atom, such Spectively, in that all of the esters have a nega 60 may usually be expelled from the molecule by tive substituent on the carbon atom alpha to the gentle heating. Type 2 condensations are gen esterified carboxyl group and for this reason erally carried out according to known considera exhibit the same general type of reactivity. Thus tions for a Knoevenagel type of reaction. This the alkylation of the nitroacetic acid ester may Condensation type is further illustrated by Ex be carried out in a manner somewhat similar to 65 ample 7 and by the illustrative reactions outlined the alkylation of malonic or cyanacetic esters and herein. Type 3 alkylations, in which the alkylat likewise Somewhat similar to the alkylations of ing agent contains either an activated double nitroparaffins, for example, with the employment bond, an epoxide system, or a thioepoxide sys of such types of compounds as mentioned in the tem, is usually carried out in the presence of a foregoing paragraph. Any suitable agent may 70 metal alkoxide, such as sodium ethoxide, or other be employed for the alkylation of the nitroace similar catalyst. tates, provided, of course, that the desired sub The nitroacetate or nitroacetate salt employed stituted nitroacetate be produced by the alkyla in the process may also be varied widely, and tion reaction, but the above-mentioned types of 75 representative esters are the methyl, ethyl, pro 2,570,29? 8 pyl, isopropyl, butyl, amyl, n-hexyl, octyl, caproyl, tivated charcoal, filtration, and several recrystal stearyl, cyclohexyl, benzyl, et cetera. Any ester lisations, amino acid crystals of exceptional or salt of an ester of the formula purity may in some instances be obtained. The NO2-CH-COOR reduction may also be carried out chemically with the employment of iron, iron flings, tin, zinc and wherein R is a hydrocarbon radical, is used in concentrated hydrochloric acid, or with (NH4)2S, the alkylation and, as in the following illustra as well as other chemical reducing agents, or in tive examples, a particular nitroacetate is en any other manner to reduce the nitro group to ployed merely as a matter of convenience. the amino group and to allow production of the The reduction of a selected nitro ester may O desired alpha amino acid. also be conducted in any suitable manner. For The method of our invention, in its broader example, in the preparation of tryptophane, we sense, contemplates the preparation of all the may place ethyl alpha-nitro-beta- (3-indole) pro naturally occurring and other primary alpha pionate, a small amount of Organic solvent, such amino acids as indicated by the following illustra as absolute ethanol or butanol, and a quantity of 15 tions, the acids produced, of course, being in each Raney nickel catalyst in an autoclave under an case of the dil form: original hydrogen pressure of 1500 p.s. i. at about Reactions of Type 2 may be illustrated as foll 25 degrees centigrade and raise the temperature OWS: to about 100 degrees centigrade until the theoreti I. For isoleucine cal amount of hydrogen is absorbed. Other hy 20 catalyst, acidic drogenation catalysts, such as Adam's catalyst, CH-CH-C=O -- HCH-COR mam PtO2, platinum, or palladium, may also be em basic or neutra ployed, and the apparatus and exact method of H NO conducting the hydrogenation reaction is of con (H) cern only in that the nitro group of the nitro 25 C-C-C=C-COR.h, lo, --'' CH-C-C-C-COsbh, th, ester be reduced to the amino group by the proce- - dure. II. For valine Likewise, if it is desired to conduct the reduc- catalyst tion and hydrolysis steps in a single operation, CH-C=O -- HCH-COR - - this too may be done. 30 &B, o, After the hydrogenation reaction, the catalyst (H) may be removed by filtration, the remaining OH3-C=C-COR. --- CH-C-C-CO liquid concentrated under vacuum, treated with &H, io, HO &H, iH, dilute sodium hydroxide solution, decolorised with activated charcoal, filtered, and adjusted 5 III. For phenylalanine

(a) (basic catalyst CH-CH=O -- HCH-COR e catalyst) o, piperidine, etc. (b)

CH-CH=O -- CH-COR (p-CH-CH-SOH)acidic catalyst CH-CH=C-COR (HD,O C-C-C-CO NO NO N toafter the thedesired desired pH withamino glacial acid usuallyacetic acid, crystallises where- (a) IV. For threonine from solution. Other conventional methods may . catalyst beacid used from to theconcentrate reaction product and crystallise of the hydrogena- the amino co-ot-coirNO tion, and methods known in the prior art will be 50 (H) found satisfactory for this purpose. Upon fur- C-C-C-COR - CH-C-C-COO ther treatment with an additional amount of ac- bH No, HO &H NH,

(b) catalyst RON CH-CH=O -- CH-COR. --> C-CH=C-COR - CH-CH-CH-COR -(H) - - CH-CH-CH-COR NO NO Ör NO &R NH, ho CH-CH-C-COB BO bR. H, acid eld CH-E-E-cottba N son 2,870,297 7 8 W. For norleucine- ates of the amino acids are indicated schematical catalyst ly by the following: cit-ch-0 -- high-c O Valine (alpha-amino-beta-methylbutyric acid)- NO 5 Intermediates: -- (H) - Alpha-nitro-beta-methylbutyric acid and CH-CH=-cNO OR Hao CH-CH.-E.NH CO Alpha-aminO-beta-methyl-butene-2-oicesters thereof. VI. For hydroxyglutamic acid- acid and esters thereof. (a) COR COR COR COR catalyst RON (H) H-CH=O -- HCH-COR. --> H-CH-i-C OR - H-E-F-C OR al-) H-E-E-coir o, NO br NO &R NE

CO to bH-CH-CH-c O O acid R. N.

COB held &H-E-F-cottÖH. NE b) catalyst Alpha-amino-beta-hydroxy-beta-meth ROC-CB-CH=0 -- figh-c OR - ylbutyric acid and esters thereof. NO Leucine (alpha - amino - gamma or methylvaleric COR H E3 COR B 30 acid)- &H.-E.--corbH NO H.-E.--C(bH NH O Intermediates:Alpha-nitro-gamma-methylvaleric acid VII. For tyrosine- and esters thereof.

(a) piperidine, etc. p-RO-CH-CH=0 -- HCH-COR basic catalyst (b) NO (p-CH-CH-SOH) (H) p-RO-CH-CH=O -- HCH-COR idle catalyst p-ro-CH-CH-i-C OR. --> pro-CH-CH-E-cor o, C y NO Y- NH H2SH) Ho pro-CH-CH-H-coh NE acid p-Ho-CH-CH-E-c OH NH VII. For Serine- Alpha-nitro-gamma-methyl - pentener catalyst 2-oic acid and esters thereof. CH=O -- HH-coir - 55 Alpha-nitro-beta-hydroxyisocaproic acid NO and esters thereof (Example 7 herein Ho-oh-fil-cotR BIOis Ho-CH-E-coat Isoleucine after).(alpha-amino-beta-methylvaleric - NO NH go acid)- N. In the above illustrative examples of type 2 Intermediates: alkylation as applied in the preparation of sev- Alpha-nitro-beta-methylvaleric acid and eral alpha amino acids from an ester of nitro- esters thereof. acetic acid, R, indicates a hydrocarbon radical, Alpha-nitro-beta-methyl-pentene-2-oic preferably an alkyl radical. Catalysts employed 65 acid and esters thereof. for the condensation step may be either acid, Alpha-nitro-beta-hydroxys beta - meth neutral, or basic, and as representative catalysts yvaleric and acid and esters thereof. are mentioned para-toluenesulfonic acid and Serine (alpha - amino - beta-hydroxypropionic piperidine, although numerous others may be emi- 9 y ypIOp. ployed with equal facility. The radical desired 70 "Etermediates: tomust, be asintroduced will be noted, into havethe nitroacetateeither an aldehyde molecule or Alphpha-nitro-beta-hydroxyproplonic it. beta-hydr ioni acid ketone group present in the molecule in order to and esters thereof. undergo the Knoevenagel type of condensation. Alpha-nitro-beta-alkoxypropionic acids The nitro acid and nitro acid ester intermedi- 75 and esters thereof. a,670,297 9 10 Threonine (alpha-amino-beta-hydroxy-n-butyric After filtration and concentration, the remain acid)- ing oil was treated in either of the following two Intermediates: ways, depending tupon the structure and water Alpha-nitro-beta-hydroxy - n - butyric Ebility of the alcohol used in the esterifica acid and esters thereof. 5 tion. Alpha-nitro-beta-alkoxy-n-butyric acids The methyl and isopropyl esters of nitroaceti and esters thereof. acid were subjected to one distillation which Alpha-nitro-beta-hydroxy-n-butene - 2 yielded the essentially pure ester. oic acid and esters thereof. The n-hexyl, cyclohexyl, and benzyl esters were Alpha-nitro-beta-alkoxy-n - buttene - 2 10 found to form sodium salts which were relatively oic acid and esters thereof. insoluble in water, insoluble in acetone and ether, Norleucine (alpha-amino-caproic acid)- and very. Soluble in alcohol. This was advanta Intermediates: Alpha-nitro-caproic acid and geous, inasmuch as the corresponding alcohols esters thereof. appear to have a low water-solubility and are not Alpha-nitro-hexene-2-oic acid and es ls easily removed from the ester product. The so ters thereof. dium Salts were isolated and purified in the foll Alpha-nitro-beta-hydroxyCaproic acid lowing manner: and esters thereof. After the removal of as much alcohol as possi Hydroxyglutamic acid (alpha-amino-beta-hy ble by distillation, the crude ester was treated, droxyglutaric acid)- 20 slowly, with stirring and cooling in an ice bath, Intermediates: Alpha-nitro-beta-hydroxy with between about 100, and 150 milliliters of 10 glutaric acid and esters thereof. per cent aqueous sodium hydroxide solution. The Alpha-nitro-beta-alkoxyglutaric acids Sodium salt which precipitated was filtered and and esters thereof. Washed twice with about fifteen milliliters of ice Phenylalanine (alpha-amino-beta-phenylpropi- 25 water and then with acetone. The salt was dried, Onic acid)- - weighed, and treated with two equivalents of hy Intermediates: Alpha-nitro-beta-phenylpro drochloric acid in about 100 milliliters of water. pionic acid and esters thereof. The reaction was generally found to be slow, and Alpha - nitro - beta - phenyl - propene prolonged shaking was usually necessary to com 2-oic acid and esters thereof. O pletely free the ester. Ether extraction, followed Alpha - nitro - beta - hydroxy - beta by Washing, drying, and concentration of the phenylpropionic acid and esters ether, resulted in the production of a relatively thereof. pure ester, as will be apparent from the analysis Tyrosine (alpha-amino-beta-para-hydroxyphen of benzyl nitroacetate obtained by the above yl propionic acid)- 35 method. Intermediates: Alpha-nitro-beta-para-hy droxyphenylpropionic acid and esters thereof, . C N Alpha - nitro - beta - para - alkoxy Theory------55.3 4.64 7.75 phenylpropionic acids and esters Found.------55.82, 4.43 7.78 thereof. 40 55.96 4.34 7.69 Alpha - nitro - beta - para - hydroxy phenylpropene-2-oic acid and esters In the preparation of the benzyl ester, it was thereof. found advantageous to use dry hydrochloric acid Alpha - nitro - beta - para - alkoxy gas as a condensing agent, and the procedure em phenylpropene-2-oic acids and esters 45 ployed was as follows: thereof. Benzyl alcohol (127 grams) and 26.3, grams of In addition to the above .intermediates, the nitroacetic acid were mixed, and dry HCl gas nitro acids and esters thereof, there is also passed into the mixture with cooling until the formed in the preparation of any amino acid by Solution was saturated at zero degrees centigrade. our new synthesis an ester of the desired amino 50 The solution was allowed to warm to room tem acid. These esters, and their formulae, will be perature and was thereafter allowed to stand for at once apparent from an inspection of the var two days. Most of the HCl gas, some benzyl ious reactions outlined hereinbefore. chloride, and some alcohol was removed by con The following detailed examples illustrate fur : centration in vacuo. After dilution with ether, ther the manner in which the principle of Our 55 washing with water, drying the ether solution, invention may be applied, but are not to be con and concentrating, the ester was treated with so strued as limiting. above.dium hydroxide and worked up as described Eacample 1.-Esters of nitroacetic acid Properties of Some representative esters of ni The following procedure was employed for the 60 troacetic acid are as follows: preparation of methyl, isopropyl, n-hexyl, and cyclohexyl esters of nitroacetic acid. Esters B. P., C. N23-269c. One mole of alcohol was introduced slowly into. Methvl ------68.515.0 mm 1.42257 . 0.38 mole of concentrated sulfuric acid with cool 65 Ethyl------F65.0 mm 1 420 subro y----- 4203 ing. Nitroacetic acid (0.25 mole) was then added n-Hexy ----- 1. 4364 at between zero and five degrees centigrade and Cyclinhexyl 1,4640 the mixture stirred until solution was obtained. ey------1.5202 The solution was then allowed to stand at room temperature for from 20 to 40 hours, at the end 70 Eacample 2.-Preparation of some esters of alpha of which time it was diluted with ice and ice nitro-beta- (3-indole) propionic acid water. Water was separated and the organic The particular nitroacetic acid ester (0.1 mole), layer diluted with two volumes of ether, after gramine (0.1 mole), and 100 milliliters of dry which the ether Solution was washed three times Xylene were placed in a three-neck flask of 250 with water and dried over magnesium sulfate. 75 milliliter capacity, which was fitted with a stirrer, 2,570,297 11 12 nitrogen inlet, thermometer, and condenser. As lution was filtered from a small amount of crys nitrogen was passed through, the contents of the talline solid ind the xylene removed by COncen flask were heated to 90-95 degrees centigrade tration in vacuo. The residual gum was dis with efficient stirring. The heating and stirring solved in chloroform and the resulting solution were continued until evolution of dimethylamine extracted twice with 50-milliliter portions of 10 from the reaction ceased, the time required usual per cent HCl and washed with water until neu ly being between 3.5 and 5 hours. The Xylene tral. The chloroform Solution was dried over was then removed in vacuo, the last traces being MgSO4 and concentracted in vacuo, whereafter removed between about 60-70 degrees centlgrade excess ethyl nitroacetate was distilled off under. at 0.5 millimeter of mercury preSSure. 10 reduced pressure, 2.9 grams coming over at 64 The crude methyl, ethyl, isopropyl, n-hexyl, degrees centigrade under 3 millimeters of mer cyclohexyl, and benzyl esters of alpha-nitro cury pressure. The residue was dissolved in beta- (3-indole)-propionic acid produced in this chloroform and extracted to exhaustion with a 5 manner were capable of further purification and per cent aqueous Solution of sodium hydroxide. identification, or could be used directly in the re The aqueous alkaline extract was then acidified duction step of the process. with 10 per cent HCl and extracted with chloro form; the extract was dried and concentrated Eacample 3-Reduction of alpha-nitro-beta and crystallisation of the remaining oil allowed (3-indole) propionic acid esters to occur. The yield of ethyl alpha-nitro-beta The ester (0.01 mole) was dissolved in about 20 (3-indole) propionate was 11.8 grams or 90 per 15 milliliters of absolute alcohol and placed in cent of theory. The melting point was 60.0 to a bomb of 40 milliliters void with about one grain 62.1 degrees centigrade. of Raney nickel catalyst. Hydrogen was intro An analytical Sample, recrystallised four times duced into the bomb up to a pressure of about . from a benzene-petroleum ether mixture, melted 2500 p.s. i., the temperature being raised to 100 25 at 62.0 to 62.8 degrees centigrade (uncorrected) degrees, centigrade as rapidly as possible with and analysed as follows: r constant rocking of the autoclave. Reduction usually occurred in about 1.5 to 2 hours, after C H. N which the bomb was allowed to cool to room tem perature, and the catalyst removed by filtration 30 Theory------59.69 5.38 10.69 and washed with alcohol. Alcohol was removed Found------59.65 5.46 10.78 by concentration, the crude ester hydrolysed by 59.62 5, 24 0.57 refluxing for 2 to 4 hours with 15 milliliters of 10 per cent aqueous sodium hydroxide, treated with Eacample 5-Preparation of tryptophane 250 milligrams of charcoal, filtered, and the solu tion brought to a pH of about 5.95 with glacial 35 Ethyl alpha-nitro-beta- (3-indole) propionate acetic acid. After removal of a Small amount of (2.62 grams), 15 milliliters of absolute ethanol dark brown amorphous material which usually and about one-half gram of Raney nickel cata precipitated immediately, the tryptophane came lyst were placed in a bomb of 40 milliliters ca out upon standing overnight in the cold. The pacity. The reduction was carried out at 100 de tryptophane was then filtered, Washed with a 40 grees centigrade and with an original hydrogen little cold water, alcohol, and finally with ether. preSSure of 1500 p.s. i. at 25 degrees centigrade. The product at this point usually had a grayish The theoretical amount of hydrogen was ab white appearance and was recrystallised from 33 Sorbed during a 12-hour period. After filtering per cent alcohol according to usual procedure. off catalyst, the alcohol was removed by concen Melting points and mixed melting points with an 45 tration under vacuum. The light colored oil which remained was heated under reflux with 16 authentic sample proved that the product from milliliters of 10 per cent aqueous sodium hydrox each of the esters was dl-tryptophane. v ide for two hours. After treating with activated charcoal (Darco G-60) and filtering, the pH of dl-Tryptophane 1 50 the solution was adjusted to 5.9 with glacial Starting Ester acetic acid. Crystals appeared, the flask was cooled in the icebox for a period of 15 hours, M.P., C.M.M.P., whereafter tryptophane was filtered off and Methyl ------.------284 285 washed with a small Ouantity of cold water. The sorronyl--- - 292 29 55 wet cake was dissolved in 100 milliliters of 33 n-Hexy 278.5 273 Cyclohexy - 275 276 rer cent alcohol, treated with an additional quan

27 titv of Darco G-60, filtered hot and allowed to Benzyl------272 cool slowly with rubbing. The tryptophane Uncorrected, decomposition. Melting point and mixed melting crystallised out in glistening plates, was filtered point taken together. 60 and washed with alcohol and with ether. The crystals were then dried in a vacuum. Eacample 4-Ethyl alpha-nitro-beta- (3-indole)- An analytical Sample was prepared by re propionate crystallising 4 times from 33 per cent alcohol. Gramine, 3-dimethylaminomethylindole (8.66 The sample melted at 278.0 to 280.0 degrees cen grams), 13.3 grams of ethyl nitroacetate and 50 tigrade and analysed as follows: milliliters of dry xylene were introduced into a 250-milliliter, three-neck flask fitted with stirrer, C H thermometer, nitrogen inlet and condenser. The temperature of the mixture was then raised to Theorv------64.69 5.92 between about 90 and 100 degrees centigrade and 70 Found------64.50 6.08 maintained at this level for about 5 hours while 64.65 5,92 passing a slow stream of nitrogen through the vigorously stirred mixture. During this time The mixed melting point with an authentic dil about one-half of the theoretical amount of di tryptophane was 278.0-280.0 degrees centigrade methylamine was evolved, whereafter the hot so 75 (uncorrected) (decompqsition). 2,570,297 13 14 Eacample 6-Preparation of tryptophane Inasmuch as various modifications may be The reduction can also be carried out chemi made in carrying out the method of the invention cally as follows: Ethyl alpha-nitro-beta- (3-in without departing from the spirit or scope there dole) propionate (2.62 grams), 3.5 grams of pow of, it is to be understood that we limit ourselves dered iron (or iron filings), 7.5 milliliters of only as defined by the appended claims. water, 7.5 milliliters of alcohol and 10 milliliters We claim: of concentrated hydrochloric acid were placed in 1. A process for the production of threonine, a 250-milliliter flask, stirred vigorously for 15 which comprises the condensation of a mono minutes and then heated, with stirring, to boiling alkyl ester of nitroacetic acid with acetaldehyde over a period of about 15 minutes. The reac O to produce an ester of alpha-nitro-beta-hydroxy tion mixture was refluxed for 15 minutes, cooled n-butyric acid, reducing the said ester with hy to room temperature, made alkaline with 10 per drogen and a hydrogenation catalyst to produce cent aqueous sodium hydroxide, filtered and the corresponding aminoacetic acid ester, hy washed with water. The clear alkaline Solution drolyzing the said ester with an aqueous alkaline was brought to a pH of 6.0 with concentrated hydrolyzing agent, and recovering threonine sulfuric acid. After seeding and rubbing, crys from the resulting hydrolysis products. , tallisation began. When crystallisation was 2. A process for the production of leucine complete the product was filtered and recrystal which comprises the condensation of a mono lised from 33 per cent alcohol, whereafter the alkyl ester of nitroacetic acid with isobutyralde d1-tryptophane melted at 283-4 degrees centi 20 hyde to produce an este' of alpha-nitro-gamma grade (uncorrected) (decomposed). A mixed methylpentene-2-oic acid, reducing the said ester melting point with an authentic dl-tryptophane with hydrogen and a hydrogenation catalyst to was 283-283.5 degrees centigrade (decomposi produce the corresponding amino acid ester, tion) (uncorrected). hydrolyzing the said ester with an aqueous al 25 kaline hydrolyzing agent and recovering leucine Eacample 7-Condensation of nitroacetic acid from the resulting hydrolysis products. esters with aldehydes 3. A process for the production of an alpha Ethyl nitroacetate (0.3 mole) and isobutyralde substituted aminoacetic acid, which comprises: hyde (0.3 mole) were mixed thoroughly and a Knoevenagel-type condensation of a monoalkyl cooled to zero degrees centigrade. Piperidine 30 ester of a nitroacetic acid with an aliphatic alde (1.5 grams) was added slowly and with stirring, hyde selected from the group consisting of acet whereafter the reaction flask was maintained at aldehyde and isobutyraldehyde to form a mono ice-temperature for two days. After dilution of alkyl ester of an alpha- (aliphatic-substituted) - the reaction product with 150 milliliters of ben nitroacetic acid, reducing the nitro group of said Zene, it was washed with 200 milliliters of dilute ester with hydrogen arid a hydrogenation catalyst hydrochloric acid and then twice with 200-milli to produce the corresponding monoalkyl amino liter portions of water. The benzene solution was acetic acid ester, hydrolyzing the said ester with dried over magnesium Sulfate and concentrated an aqueous alkaline hydrolyzing agent, and re in vacuum to a quantity of 54.6 grams. This covering the alpha-substituted aminoacetic acid residual liquid was distilled in a vacuum using a 40 from the resulting hydrolysis products. magnetic stirrer in the distillation flask, which DAWD ... WEISBLAT. Speeded the distillation and allowed a lower DOUGLAS A. LYTITLE. bath temperature. Four cuts were made, the main fraction being the third, which boiled from REFERENCES CITED 69 degrees centigrade at 0.35 millimeter to 82 degrees centigrade at 0.32 millimeter of mercury The following references are of record in the pressure. The weight of this cut was 28.53 grams, file of this patent: a yield of 46.3 per cent of theory. The refractive UNITED STATES PATENTS index of the cut at 27 degrees centigrade was Number Name Date 1.4442. The fraction was redistilled and four 2,005,538 Engelmann ------June 18, 1935 cuts made to obtain an analytical sample. The 2,071,327 Bley ------Feb. 23, 1937 fourth cut analysed as follows: 2,152,852 Loder ------Apr. 4, 1939 2,301,829 Studer ------Nov. 10, 1942 C H N 2,334,163 Kirk ------Nov. 16, 1943 55 2,380,479 Stiller ------July 31, 1945 Theory for ethylalpha-nitro-beta-hydroxy is0ciproate.------46.82 7.37 6.83 OTHER REFERENCES Found ------. 46.88 7.34 6.78 Bouveault: Bull. Soc. Chim. de Paris, 3rd Series, vol. 25 (1901), pp. 911-914. This compound, ethyl alpha-nitro-beta-hy Wahl: Bull. Soc. Chim. de Paris, 3rd series, vol. droxyisocaproate, is the intermediate for leucine. 25 (1901) pp. 918-929. in a similar manner, acetaldehyde is con Jackson: J. Am. Chem. Soc., vol. 52 (1930) pp. densed with a nitroacetate to yield the threonine 5029-5035. intermediates, esters of alpha-nitro-beta-hy Beilstein, 4th ed., 2nd suppl., vol. 2 (1942); p. droxy-n-butyric acid. 65 234, citing: Steinkopf, Annalen, vol. 434, p. 29. Reference is made to our copending application Albertson: J. Am. Chem. Soc., vol. 67 (1945), Serial No. 733,982, filed March 11, 1947, now pp. 36-37. Patent No. 2,557,041, in which is claimed subject Lynn: Organic Chem. (Lea and Febiger, 3rd matter disclosed or originally included but not ed.), 37, 195 (1948). claimed herein. 70